Ágnes Rimanóczy

Modulation of catecholamine turnover rate in brain regions of rats exposed prenatally to morphine

The concentration and turnover rate of brain catecholamines were measured in the hypothalamus, preoptic area (POA), frontal cortex, striatum and cerebellum of adult male and female rats exposed in utero to morphine (5-10 mg/kg/twice a day) during gestation days 11-18. Norepinephrine (NE) and dopamine (DA) turnover rates were estimated following alpha-methylparatyrosine (AMPT) administration. Prenatal morphine altered NE content and turnover in male and female rats in a regionally specific, sexually dimorphic manner. Basal NE content increased approximately 60% in the hypothalamus of male rats, but it decreased about 30% in the hypothalamus of female rats. NE turnover in the hypothalamus of morphine-exposed rats increased 50% in males and decreased 50% in females. Prenatal morphine had no effects on NE turnover in the male POA, but in female rats NE turnover decreased approximately 60%. Alterations in the frontal cortex of morphine-exposed male and female rats resembled the pattern in the hypothalamus; however, the differences did not reach statistical significance. In addition, prenatal morphine had no effect on striatal or cerebellar NE or on basal levels or turnover of DA in any brain regions. These results demonstrate that prenatal morphine alters the content and turnover of NE in a sexually dimorphic manner in specific brain regions of male and female rats, suggesting alterations in the density of terminals and/or utilization of NE. These sexually dimorphic alterations in hypothalamic NE induced by prenatal morphine may be related to the changes observed in adult male and female sexual behavior in our previous work.

Prenatal exposure to morphine differentially alters gonadal hormone regulation of δ-opioid receptor binding in male and female rats

The present study tested the hypothesis that exposure to morphine on gestation days 11-18 differentially alters delta-opioid receptors in the brain of adult male and female rats. In Experiment 1, the binding characteristics of delta-opioid receptors were examined in membrane homogenates from six brain regions, including the hypothalamus (HYP), preoptic area, frontal cortex (CX), ventral tegmental area, striatum (STR) and cerebellum of adult male and female rats. In Experiment 2, the density of delta-opioid receptors was assessed in the CX and STR using receptor autoradiography. Prenatal morphine exposure has no effects on delta-opioid receptors in the brain of gonadally intact, adult male rats regardless of methodology. However, when male rats were gonadectomized in Experiment 2, morphine-exposed males have fewer delta-opioid receptors than controls in the CX but not in the STR. These reductions in cortical delta-opioid receptors are restored by testosterone replacement, demonstrating that prenatal morphine exposure alters testosterone regulation in the CX of male rats. In ovariectomized (OVX) female rats, prenatal morphine exposure increases the density of delta-opioid receptors in the frontal CX. Interestingly, this up-regulation of delta-opioid receptors is not present when the CX is investigated by autoradiography. Moreover, progesterone given alone or in combination with estrogen reduces the density of delta-opioid receptors in the CX and STR of both saline- and morphine-exposed, OVX females. Thus, mid to late gestational morphine exposure differentially alters the influence of adult gonadal hormones on delta-opioid receptors in the CX, decreasing the sensitivity in females and increasing it in males. This is also the first report to demonstrate that gonadal hormones regulate delta receptor densities in brain regions other than the HYP of OVX females.

Autoradiographic evidence that prenatal morphine exposure sex-dependently alters μ-opioid receptor densities in brain regions that are involved in the control of drug abuse and other motivated behaviors

The present study examined the effects of prenatal morphine exposure on mu-opioid receptor density in young adult male and female rats to assess the long-term alterations in several brain areas including the nucleus accumbens (NAc), bed nucleus of stria terminalis (BNST), and the basolateral (BLA), lateral (LA), central (CeA), and posteromedial cortical (PMCoA) amygdaloid nuclei. These brain areas are involved in motivating and rewarding behaviors of opiates and other drugs of abuse. The reinforcing actions of opiates appear to be mu-opioid receptor dependent. The results demonstrate that in male rats, prenatal morphine exposure significantly increases the density of mu-opioid receptors in the NAc and PMCoA. In contrast, the same prenatal morphine exposure reduces the density of mu-opioid receptors in the BLA, while increasing it in the CeA and without effects in the LA or BNST. In female rats, prenatal morphine exposure has no effects on the density of mu-opioid receptors in the above six brain areas, but the density of these receptors is dependent on the presence or absence of ovarian hormones. Thus, the present study demonstrates that mid- to late gestational morphine exposure induces long-term, sex-specific alterations in the density of mu-opioid receptors in the NAc and amygdala. Moreover, this prenatal morphine exposure also eliminates sex differences in the density of mu-opioid receptors in the NAc, CeA, and PMCoA but not in the BLA, LA, and BNST.

Adrenocorticotropin Stress Response but Not Glucocorticoid-Negative Feedback Is Altered by Prenatal Morphine Exposure in Adult Male Rats

The present study was designed to investigate the effects of prenatal morphine exposure on the hypothalamic-pituitary-adrenal (HPA) axis-regulated stress responses by measuring restraint stress-induced changes in the adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels. In experiment 1, plasma levels of ACTH and CORT in prenatally morphine-, saline-exposed and control male rats were determined before and at several times after restraint stress. There were no statistically significant differences in plasma ACTH and CORT levels before restraint stress between the groups. However, prenatal morphine exposure dampened the stress-induced increase and spontaneous recovery of ACTH levels after the restraint stress. There were no differences in plasma CORT levels between the three groups either before or at any time after restraint stress. Experiment 2 was designed to investigate the sensitivity of negative feedback of glucocorticoids using the dexamethasone (DEX) suppression test. DEX was administered at different doses (0.001, 0.01, 0.1 and 1.0 mg/kg) and ACTH and CORT plasma levels were measured before and at several times after restraint stress in prenatally morphine- and saline-exposed males. DEX pretreatment eliminated the differences observed in ACTH responses to stress in morphine- and saline-exposed males. DEX pretreatment dose dependently suppressed the restraint stress-induced increased plasma ACTH concentration. In plasma CORT levels, DEX pretreatment dose dependently suppressed the restraint stress-induced increased plasma CORT concentration regardless of prenatal drug exposure. Thus, the present study demonstrates that prenatal morphine exposure alters the ACTH and CORT responses to stress but not the sensitivity of negative feedback of glucocorticoids.

Field-specific changes in hippocampal opioid mRNA, peptides, and receptors due to prenatal morphine exposure in adult male rats.

Alterations in the opioid system in the hippocampal formation and some of the possible functional consequences were investigated in adult male rats that were prenatally exposed to either saline or morphine (10 mg/kg twice daily on gestational days 11-18). In situ hybridization and Northern blots were used to measure proenkephalin and prodynorphin mRNA, and radioimmunoassays quantified proenkephalin- and prodynorphin-derived peptide levels in the dentate gyrus, CA3, and CA1 subfields of the hippocampal formation. Prenatal morphine exposure in male rats decreases proenkephalin and increases prodynorphin mRNA selectively in the granule cell layer of the dentate gyrus. Similarly, met-enkephalin peptide levels are decreased and dynorphin B peptide levels are increased in the dentate gyrus but not CA3 or CA1 of prenatally morphine-exposed males. In addition, there are decreases in dynorphin-derived peptides in the CA3 subfield. Receptor autoradiography revealed increases in the density of micro but not delta receptor labeling in discrete strata of specific hippocampal subfields in morphine-exposed males. Because alterations in the hippocampal opioid system suggest possible alterations in the excitability of the hippocampal formation, changes in opioid regulation of seizures were examined. Morphine exposure, however, does not alter the latency to onset or number of episodes of wet dog shakes or clonic seizures induced by infusion of 10 nmol [D-Ala2, MePhe4, Gly-ol5]enkephalin into the ventral hippocampal formation. Interestingly, a naloxone (5 mg/kg) injection 30 min before bicuculline administration reverses the increased latency to onset of clonic and tonic-clonic seizures in morphine-exposed males. Thus, the present study suggests that exposure of rats to morphine during early development alters the hippocampal opioid system, suggesting possible consequences for hippocampal-mediated functions.

Prenatal morphine exposure alters estrogen regulation of κ receptors in the cortex and POA of adult female rats but has no effects on these receptors in adult male rats

The binding characteristics of kappa receptors were assessed in the frontal cortex (CX), striatum, hypothalamus, preoptic area (POA), cerebellum, and ventral tegmental area of adult male and female rats exposed prenatally to morphine or saline. Prenatal morphine exposure altered estrogen regulation of kappa receptors in the CX and POA of females, but had no effects on kappa receptors in any of the examined brain regions in male rats.

Sex dimorphic alterations in postnatal brain catecholarnines after gestational morphine

The concentration of brain catecholamines was measured in the hypothalamus, preoptic area (POA), frontal cortex, cerebellum, and striatum of rats exposed in utero to morphine (5-10 mg/kg/twice daily) during gestation days 11-18. Prenatal morphine induced regionally specific, sexually dimorphic alterations in male and female norepinephrine (NE), and dopamine (DA) content at different postnatal ages. Prenatal morphine significantly increased NE content in the hypothalamus of both sexes at postnatal day (PND) 23. In the POA, on the other hand, morphine increased NE content in exposed males at PND 23 and in females at PND 33. In the cerebellum, the NE content of both sexes was significantly elevated at PND 45. In the striatum, NE content was increased by the prenatal morphine only in females at PND 16. The concentration of DA was also affected in a sexually dimorphic manner. At PND 16, prenatal morphine increased the levels of hypothalamic DA only in males, and it reduced the content of DA in female but not male POA. At PND 45, prenatal morphine increased DA in the hypothalamus of females and decreased it in males. In the cerebellum of 16-day-old morphine-exposed animals, DA levels were increased only in males; at PND 45, the levels of DA were still increased in males but had not changed in females. In the striatum, the DA content was reduced only in males at PND 16. Thus, prenatal morphine alters the development of both NE and DA neurotransmitter systems in the hypothalamus, POA, striatum, and cerebellum in a sexually dimorphic manner.

Hypothalamo-Pituitary-Adrenal Axis-Regulated Stress Response and Negative Feedback Sensitivity Is Altered by Prenatal Morphine Exposure in Adult Female Rats

It has been shown that adult female rats react to stressors more intensely than adult male rats. Our previous work demonstrated that the adrenocorticotropic hormone (ACTH) but not corticosterone (CORT) response to stress is altered by prenatal morphine exposure in adult male rats. Response of the hypothalamo-pituitary-adrenal (HPA) axis to stress is known to be sex specific and dependent on the hormonal fluctuation of the estrous cycle. Therefore, the present study examined the effect of prenatal morphine exposure on the levels of ACTH and CORT before and after restraint stress in adult female rats. Experiment 1 tested ACTH and CORT plasma levels before and after restraint stress in prenatally morphine- and saline-exposed, adult diestrus and proestrus female rats. Prenatal morphine exposure suppressed the restraint stress-induced ACTH levels in both diestrus and proestrus females, but did not have any effects on the basal or stress-induced CORT levels. Experiment 2 examined the sensitivity of negative feedback using the dexamethasone (DEX) (0.001, 0.01, 0.1 and 1.0 mg/kg) suppression test in adult, prenatally morphine- and saline-exposed female rats. In saline-exposed, proestrus but not diestrus females, all doses of DEX were effective in suppressing the restraint stress-induced increase in CORT levels. In both diestrus and proestrus, morphine-exposed females, only the two highest doses of DEX (0.1 and 1.0 mg/kg) were successfully suppressing the stress-induced CORT levels. The stress-induced increase in the ACTH level was suppressed only by the highest dose of DEX (1.0 mg/kg) in both saline- and morphine-exposed, diestrus and proestrus females. Thus, the present study demonstrates that prenatal morphine exposure alters the HPA axis-regulated stress response and the sensitivity of negative feedback that are affected by the fluctuation of ovarian hormones.

Mu-opioid receptors in seizure-controlling brain structures are altered by prenatal morphine exposure and by male and female gonadal steroids in adult rats

The present study used autoradiography to examine the effect of prenatal morphine exposure on mu-opioid receptor density in epileptic seizure-controlling brain structures including the substantia nigra pars compacta (SNC), substantia nigra pars reticulata (SNR), superior colliculus (SC), and subthalamic nucleus (STN) of adult male and female rats. The results demonstrate that prenatal morphine exposure increases the mu-opioid receptor density in the SNC and STN, but not in the SNR or in the SC of gonadally intact adult male rats. The density of mu-opioid receptors in the SNC and STN is, however, decreased following gonadectomy in morphine-exposed males, and testosterone treatment fails to restore this decrease to the level of gonadally intact males. Further, in the SC, the density of mu receptors was lower in both saline-exposed, gonadectomized (GNX) and GNX, TP-treated males and in morphine-exposed, GNX, TP-treated males relative to gonadally intact saline- and morphine-exposed males, respectively. In ovariectomized (OVX) female rats, the same prenatal morphine exposure increases the mu-opioid receptor density in the SNC and SNR, but decreases it in the STN. The density of mu-opioid receptors is also decreased in the SNC and SC of OVX estrogen-treated females and in the SNR and SC of OVX, progesterone-treated females. Thus, the present study demonstrates that mu-opioid receptors in seizure-controlling brain structures are sex-specifically altered by prenatal morphine exposure in adult progeny. Further, prenatal morphine exposure alters gonadal hormone effects on the density of mu receptors in adult, OVX females.

Cortical and striatal μ-opioid receptors are altered by gonadal hormone treatment but not by prenatal morphine exposure in adult male and female rats

The cerebral cortex (CX), cingulate CX (cgCX), and striatum (STR) play an important role in locomotion, cognition, emotion, and reward-motivated behaviors, and are altered by prenatal morphine exposure. We have demonstrated that delta-opioid receptors in the CX and STR of adult male and female rats are altered by prenatal morphine exposure and gonadal hormonal treatment. Because morphine binds with greater affinity to mu- than delta-opioid receptors, the present study examined the effect of prenatal morphine exposure on mu-opioid receptor density in the CX, cgCX, and STR of adult male and female rats using receptor autoradiography. In Experiment 1, three groups of adult male rats were analyzed: intact, gonadally intact; GNX, gonadectomized; and TP, GNX and testosterone propionate (TP)-treated. In Experiment 2, four groups of adult females were analyzed: OVX, ovariectomized; EB, OVX and estradiol benzoate (EB)-treated; P, OVX and progesterone (P)-treated; and EB+P, OVX and EB- and P-treated. In male rats, GNX and TP males had lower mu-opioid receptor densities in all three brain regions than gonadally intact males regardless of prenatal drug exposure. In female rats, OVX, EB+P-treated females had lower mu-opioid receptor density in the STR than OVX only females regardless of prenatal drug exposure. There were no drug or gonadal hormone effects in the CX or in the cgCX of female rats. Thus, the present study demonstrates that gonadal hormones, and not prenatal morphine exposure, alter the density of mu-opioid receptors in the CX, cgCX, and STR of adult male and female rats.